MCB Accepts, published online ahead of print on 29 September 2014 Mol. Cell. Biol. doi:10.1128/MCB.00524-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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Paip1, an effective stimulator of translation initation, is targeted by
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WWP2 for ubiquitination and degradation
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Yanrong Lv1, Kai Zhang1, Haidong Gao1*
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Department of Breast Surgery, QiLu Hospital of Shandong University, Jinan, Shandong,
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China1
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Running title: WWP2 ubiquitylates and degrades Paip 1
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* Corresponding author. Haidong Gao, Department of Breast Surgery, QiLu Hospital of Shandong University No. 107, Wen hua Xi Road, Jinan, China, Tel: +86 15806661668, E-mail: [email protected] 1
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ABSTRACT
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Poly(A)-binding protein-interacting protein 1 (Paip1) stimulates translational initiation by
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inducing the circularization of mRNA. However, the mechanisms underlying Paip1
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regulation, particularly its protein stability, are still unclear. Here we show that the E6AP
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carboxyl terminus (HECT)-type ubiquitin ligase WW domain-containing protein 2
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(WWP2), a homolog of HECT-type ubiquitin ligase WWP1, interacts with and targets
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Paip1 for ubiquitination and proteasomal degradation. Mapping of the region, including
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the WW domain of WWP2, revealed the interaction between WWP2 and the
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PABP-binding motif 2 (PAM2) of Paip1. The two consecutive PxxY motifs in PAM2 are
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required for WWP2-mediated ubiquitination and degradation. Furthermore, ectopic
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expression of WWP2 decreases translational stimulatory activity with the degradation of
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Paip1. We therefore provide the evidence that the stability of Paip1 can be regulated by
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ubiquitin-mediated degradation; thus, highlighting the importance of WWP2 as a
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suppressor of translation.
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INTRODUCTION
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Regulation of gene expression occurs in eukaryotes during messenger RNA (mRNA)
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translation, specifically at the initiation of translation. Deregulation at this step of the
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translation process leads to abnormal gene expression, which in turn alters cell growth
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and possibly leads to cancer development (1–3). Translational initiation comprises a
36
series of discrete steps and starts with the dissociation of 80S ribosomes into subunits. 2
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The eukaryotic translational initiation factors (eIFs) 1, 1A, and 5, and the eIF3 complex
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promote the binding of the eIF2-GTP-Met-tRNAi ternary complex to the 40S subunit,
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thereby forming a 43S pre-initiation complex (PIC) (4–7). The 43S PIC is loaded onto the
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mRNA near the 5’-7-methylguanosine cap by numerous factors, including eIF3,
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poly(A)-binding protein (PABP), eIF4B, and eIF4F complex. The eIF4F complex
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comprises three subunits, namely, a cap-binding protein (eIF4E), an RNA helicase
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(eIF4A), and a scaffold protein eIF4G (8,9). eIF4G harbors the binding domains for
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PABP, eIF4E, eIF4A, and eIF3 in mammals. The binding domains for eIF4E and PABP
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in eIF4G enable the assembly of a stable, circular messenger ribonucleoprotein (mRNP)
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by eIF4G; eIF4G–eIF3 interaction generates a protein bridge between the mRNPs
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(10–13).
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PABP-interacting protein 1 (Paip1) is a PABP-binding protein that contains two
49
distinct PABP-binding motifs (PAMs). PAM1 binds to RNA recognition motif 2 in the N
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terminus of PABP; PAM2, which is a conserved region comprising approximately 15
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amino acids, binds to the PABC domain of PABP (14, 15). Paip1 shows 39% similarity
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to eIF4G and the eIF4G-related protein, p97/DAP5/NAT1 (16–18). A specific portion
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that is present in both Paip1 and eIF4G has one of two known eIF4A binding regions and
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an eIF3 binding site (19, 20). The abovementioned findings indicate that Paip1
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co-immunoprecipitates with eIF4A and eIF3 (21, 22). The presence of Paip1 in animal
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cells may indicate the involvement of a mechanism that links PABP to eIF4A, thereby
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causing the circularization of mRNA (21). Data from previous studies suggest that the 3
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interaction of Paip1 with eIF3 stabilizes circular mRNP conformation, which is formed
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by eIF4G–PABP interaction (22). eIF3 is reportedly phosphorylated by S6K1/2, which
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stimulates the Paip1–eIF3 interaction and the initiation of translation (23). Paip1 is an
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important positive effector of translational initiation, but information is lacking on the
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mechanism underlying Paip1 regulation.
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In the present study, we demonstrated the critical function of an E6AP carboxyl
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terminus (HECT)-domain comprising an E3 ubiquitin ligase WW domain-containing
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protein 2 (WWP2), which is also known as atrophin-1 interacting protein 2 (AIP2), in the
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regulation of Paip1 protein stability. WWP2 is homologous to the HECT domain-type
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ubiquitin-protein ligase and participates in the regulation of craniofacial development and
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chondrogenesis (24, 25). WWP2 also participates in the maintenance of key oncogenic
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signaling pathways, which are linked to cancer cell growth, survival, and tumor spread
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(26, 27). Here we showed that WWP2 interacted with the PAM2 motif of Paip1 via the
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WW domain. Further investigation revealed that WWP2 targeted Paip1 for ubiquitination
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and degradation via the “PEFYPSGY” sequence in the PAM2 motif. Importantly, WWP2
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was found to participate in translational initiation by regulating Paip1 protein level.
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MATERIALS AND METHODS
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Cell Culture and Transfection. HEK293T and HeLa cells were cultured in Dulbecco’s
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modified Eagle’s medium supplemented with 10% fetal bovine serum and 5 U/ml
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penicillin-streptomycin (Gibco, USA) in 5% CO2. Cells were transfected by
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Lipofectamine 2000 following the manufacturer’s protocol (Invitrogen, USA). 4
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Antibodies and Reagents. The proteasomal inhibitors MG132 and lactacystin were
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purchased from Sigma-Aldrich, USA. The WWP2 and Paip1 antibodies were purchased
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from Abcam, UK. Anti-glyceraldehyde 3-phosphate dehydrogenase (Anti-GAPDH) and
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secondary antibodies were purchased from Santa Cruz Biotechnology, Inc., USA.
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Anti-hemagglutinin (Anti-HA) was obtained from Roche Applied Science, Germany, and
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anti-Myc and anti-Flag antibodies were from MBL.
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Immunoprecipitation and Immunoblotting. For general cell lysis, transfected cells
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were harvested and lysed in HEPES lysis buffer (containing 20 mM HEPES, pH 7.2,
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50 mM NaCl, 0.5% Triton X-100, 1 mM NaF, and 1 mM dithiothreitol) and boiled with
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2× SDS–PAGE loading buffer. For immunoprecipitation, cell lysates were prepared in
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500 ml HEPES buffer supplemented with a protease inhibitor mixture (Roche Applied
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Science). Immunoprecipitation was performed by primary antibody incubation for 3 h
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followed by overnight incubation with Protein A/G Sepharose beads (Santa Cruz). The
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beads were washed with HEPES buffer thrice and examined by immunoblotting.
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Immunofluorescence. For subcellular localization analyses, cells were fixed with 4%
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paraformaldehyde and permeabilized in 0.2% Triton X-100 (phosphate-buffered saline).
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Proteins were stained using the indicated antibodies and detected with a TRITC- or
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FITC-conjugated secondary antibody. The nuclei were stained with 4′,6′
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-diamidino-2-phenylindole hydrochloride (DAPI; Sigma), and images were visualized
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with a Zeiss LSM 510 Meta inverted confocal microscope.
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RNA Interference. The WWP2 siRNA-1 (5’- CACCTACTTTCGCTTTATA5
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3’), siRNA-2 (5’- GGAGTACGTGCGCAACTAT-3’), and non-targeting siRNAs
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(5’-UUCUCCGAACGUGUCACGU-3’) were synthesized by Shanghai GenePharm. All
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siRNAs were transfected into the cells according to the manufacturer’s protocol.
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Determination of turnover of Paip1. HEK293T cells were transfected with Myc-Paip1
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with Flag-WWP2, Flag-WWP2-C838A, or empty vector (or interfering RNA). The cells
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were treated with cycloheximide (CHX, 15 mg/ml, Sigma) at 36 h after transfection and
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harvested at the indicated time. The protein level was measured by Western blot using the
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indicated antibodies.
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Ubiquitination assays. Cells were transfected with Myc-Paip1 and Flag-WWP2 or its
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mutants to investigate Paip1 ubiquitination. Subsequently, cells were treated with MG132
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(20 ìM) for 8 h before harvest. Cell lysis solution was prepared in modified RIPA lysis
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buffer containing 10 mM Tris–HCl, pH 7.5, 150 mM NaCl, 5 mM
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ethylenediaminetetraacetic acid (EDTA), 1% NP-40, 1% sodium deoxycholate, 0.025%
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sodium dodecyl sulfate, and protease inhibitors. Immunoprecipitation was conducted
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using the indicated antibody. Subsequently, immunoblot analysis was performed. For in
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vitro ubiquitination assay, His-WWP2 and glutathione s-transferase (GST)-Paip1 were
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expressed in Escherichia coli and were purified with Ni-nitrilotriacetate-agarose (Qiagen,
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Netherlands) and glutathione-Sepharose 4B beads (Amersham, UK), respectively. The
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assays were conducted in 30 il ubiquitination assay buffer (containing 50 mM Tris, pH
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8.0, 50 mM NaCl, 1 mM dithiothreitol, 5 mM MgCl2, and 3 mM ATP) with 0.7 ìg of E1,
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1 ìg of UbcH5c (E2), 15 ìg of HA-ubiquitin (Boston Biochem, MA, USA), 0.7 ìg of 6
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His-WWP2 (wild-type or C838A mutant), and 1.5 ìg GST–Paip1. Samples were
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incubated at 30 °C for 2 h. Reactions were terminated using the sample buffer.
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Real-time reverse transcription–polymerase chain reaction (RT–PCR). Total RNA
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was isolated from the cells using TRIZOL (Invitrogen) and reverse transcribed using 1 ìg
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of total RNA with an oligo(dT) primer. The following primers were used for real-time
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PCR: human GAPDH forward,
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5’-GGGAAGGTGAAGGTCGGAGT-3’; GAPDH reverse, 5’-
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TTGAGGTCAATGAAGGGGTCA-3’; human WWP2 forward,
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5’- CGCAACTATGAGCAGTGGCA-3’; human WWP2 reverse,
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5’- GGTCGTGCGAGTGTTATGGT-3’; human Paip1 forward, 5’-
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GGAGAACTGGAAAGCCGAGGGTA-3’; and human Paip1 reverse,
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5’- GTGTAACTGGAAGAATAACCTGAAGGG-3’; Renilla forward
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5’-CAGTGGTGGGCCAGATGTAAACAA-3’; and Renilla reverse
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5’-TAATACACCGCGCTACTGGCTCAA-3’.
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Translation assays. HeLa cells were grown in 60 mm dishes and co-transfected with
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Myc-Paip1, pTet-HA-WWP2 (WT or CA) or control vector pUHD-15-1 expressing the
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Tet-controlled transactivator (tTA) (28), and pRL-CMV (Promega, USA). pRL-CMV is a
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Renilla luciferase reporter construct. The medium was replaced with Tet at a final
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concentration of 0 or 300 ng/ml after 4 h. Cells were harvested 48 h after transfection,
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and extracts were used to quantify Renilla luciferase by a dual-luciferase reporter assay
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system (Promega). Protein concentration was determined using Bio-Rad protein assay 7
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reagent, and Renilla luciferase activity was corrected according to the protein
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concentration. The relative induction for each construct was determined by calculating
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the ratio of Renilla luciferase activity between the induced condition (no Tet) and the
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repressed condition (with 300 ng/ml Tet). Extracts were subjected to SDS–PAGE and
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Western blot analysis.
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Results
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WWP2 negatively regulates the protein level of Paip1 in a proteasome-dependent
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manner. To explored the mechanism underlying Paip1 regulation, we first investigated
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whether or not the Paip1 protein was regulated through the proteasome pathway, which is
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the most critical pathway in the regulation of cellular protein stability and quality in
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eukaryotes. The level of endogenous Paip1 protein increased significantly after MG132
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treatment in both HEK293T and HeLa cells (Fig. 1A), thereby suggesting that Paip1
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protein regulation depended on the proteasome system. We also examined the effect of
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MG132 treatment on the levels of other related translation factors, such as PABP and
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Paip2. The proteasome inhibitors failed to affect the PABP and Paip2 protein levels (Fig.
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1A). The ubiquitin ligases controlled ubiquitination and determined the specificity of
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substrate recognition during ubiquitin-mediated proteasomal degradation. So we
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determined which ubiquitin ligase downregulated Paip1 protein stability. Sequence
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analysis showed that Paip1 comprised three putative PxxY-type PY motifs (Fig. 1B).
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These motifs are recognized by the Nedd4 family of ubiquitin ligases. Among the nine
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members of the Nedd4 ligases, only WWP2 specifically downregulated Paip1 protein 8
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levels (Fig. 1C). The downregulation of Paip1 by WWP2 was blocked by proteasome
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inhibitors MG132 and lactacystin (Fig. 1D). Thus, we hypothesized that WWP2 functions
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as an ubiquitin ligase in Paip1 degradation.
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To test our hypothesis, we transiently transfected HEK293T cells with a constant
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amount of Paip1 and an increasing amount of wild-type WWP2 or ligase-inactive
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WWP2-C838A expression vectors. The Paip1 protein level progressively decreased with
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increasing WWP2-WT expression, but this phenomenon was not observed with increasing
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WWP2-C838A mutant expression (Figs. 1E and 1F). No change in the Paip1 mRNA level
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was observed. Thus, WWP2 could mediate Paip1 destruction depending on its ubiquitin
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ligase activity.
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To verify whether endogenous WWP2 regulates Paip1 level, WWP2 was depleted
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using two independent siRNAs. The depletion of endogenous WWP2 in HEK293T and
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HeLa cells significantly increased Paip1 protein level (Fig. 1G). However, the mRNA
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level was not affected by the abovementioned changes (Fig. 1H). WWP2 can function as
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an ubiquitin ligase to maintain proper Paip1 protein levels in human cells.
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Paip1 and Paip2 share similar domains and compete for PABP binding. According
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to previous studies, another HECT-type ubiquitin ligase, namely, EDD, targets Paip2 for
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degradation during PABP depletion. We examined the effect of WWP2 overexpression
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on Paip2 and found that WWP2 did not affect Paip2 protein levels (Fig. 1I). Furthermore,
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overexpression of Paip2 did not affect WWP2-mediated Paip1 degradation (Fig. 1J).
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Paip1 and Paip2 did not compete for WWP2 binding, and the degradation of the two 9
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proteins required different ubiquitin ligases. Moreover, the Paip1 binding protein, PABP,
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was not regulated upon WWP2 silencing and overexpression (Fig. 1I).
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WWP2 promotes the degradation and ubiquitination of Paip1. To assessed
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whether the reduction in the protein level of Paip1, which was induced by WWP2
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overexpression, was due to increasing Paip1 degradation. We analyzed the steady-state
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levels of Paip1 by applying CHX, an inhibitor of protein synthesis. The half-life of
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exogenously expressed Paip1 was greatly reduced by the expression of WWP2-WT, but
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not by the expression of Paip1-C838 A (Fig. 2A). However, the depletion of WWP2
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prolonged the half-life of endogenous Paip1 (Fig. 2B). Thus WWP2 promoted the
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degradation of the Paip1 protein.
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We determined whether WWP2 functioned as an ubiquitin ligase and if WWP2
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directly promoted the ubiquitination of Paip1. An in vitro ubiquitination assay was
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initially conducted using purified E1 and UbcH5c, bacteria-expressed His-WWP2-WT or
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His-WWP2-C838A, and GST-Paip1 or GST (control). Bacteria-expressed and purified
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WWP2-WT promoted the ubiquitination of Paip1, but such effect on Paip1 was not
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induced by the WWP2-C838A (Fig. 2C). Overexpressed WWP2 enhanced the
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ubiquitination of Paip1 in the presence of MG132 in HEK293T cells (Fig. 2D). The
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required WW and HECT domains were sufficient for WWP2 to promote the degradation
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of Paip1 (Fig. 2E). These data suggested that WWP2 functioned as a biologically active
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ubiquitin ligase and induced the ubiquitination and degradation of Paip1.
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The WW domain of WWP2 is required for binding to the PAM2 motif of Paip1. 10
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Proteins should at least partly colocalize to be functionally linked. Indirect
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immunofluorescence assays revealed that WWP2 and Paip1 colocalized predominantly in
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the cytoplasm (Fig. 3A). We further confirmed the interaction of WWP2 with Paip1 in
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mammalian cells. A co-immunoprecipitation assay revealed the association between
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Paip1 and WWP2-WT or C838A mutant (Fig. 3B). Thus, ubiquitin ligase activity was not
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required for the interaction. In addition, endogenous Paip1 and its binding partners were
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co-immunoprecipitated with WWP2, but not with the control IgG from HeLa cells
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(Fig. 3C left panel). Given the interactions between WWP2 and eIF3a, PABP disappeared
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in a Paip1 depletion cell extract (Fig. 3C right panel), thereby suggesting that eIF3a and
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PABP bonded to WWP2 depending on the presence of Paip1.
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The direct association between WWP2 and Paip1 was confirmed by GST pull-down
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assay. WWP2 protein comprised three domains, namely, C2, WW (including four tandem
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WW domains), and HECT. The results of the GST pull-down assays indicated that the
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WW domain mediated the direct interaction between WWP2 and Paip1 (Fig. 3D). The C2
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or HECT domains of WWP2 did not show the same regulatory action. The
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abovementioned results contradicted a previous finding, which stated that the WW
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domains of Nedd4 family members preferred to recruit substrates (29). Paip1 comprised
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two binding sites for PABP, namely, PAM1 and PAM2 (15). PAM2 was required for the
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binding of Paip1 to eIF3. Different parts of Paip1 were fused to GST to identify the
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WWP2-binding site in Paip1. His-WWP2 was detected in reactions involving the
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full-length protein, the fusion protein with retained Paip1 N terminus (comprising amino 11
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acids 1 to 143), and the PAM2 motif (comprising amino acids 116 to 143). However, the
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large C-terminal region (comprising amino acids 144 to 479) was not required for the
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binding of Paip1 to WWP2 (Fig. 3E). The PAM2 motif was previously identified in
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numerous proteins with diverse functions (30), thereby suggesting that PAM2
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participated in protein–protein interactions in a wide range of cellular processes. Two of
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the three PxxY motifs were typical binding regions of the WW domains of the Nedd4
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family members (31) and were located in the PAM2 motif of Paip1 (Fig. 1B). Paip1
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interacted with PABP via the PAM2 motif. We measured the interaction of Paip1 and
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PABP upon WWP2 silencing and overexpression to determine whether or not WWP2 and
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PABP competed for Paip1 binding. WWP2 did not affect the interaction of Paip1 and
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PABP (Fig. 3F), thereby suggesting that Paip1’s PAM2 motif (comprising amino acids
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116 to 143) sufficiently interacted with the WW domain of WWP2.
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Degradation and ubiquitination of Paip1 by WWP2 depends on the presence of
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the PEFYPSGY sequence. Based on data from previous studies, we speculated that the
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two PxxY motifs (PEFYPSGY sequence) in PAM2 participated in WWP2’s recognition
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and degradation of Paip1. We transfected different Paip1 deletion mutants into HEK293T
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cells with Flag-WWP2-WT to determine the importance of the two PxxY motifs.
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Degradation analysis showed that mutants 144 to 479 included one PxxY motif that was
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not degraded by overexpressed WWP2. By contrast, the N-terminal mutants 1 to 143,
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which comprised the first two PxxY motifs, were efficiently degraded by WWP2 (Fig.
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4A). This phenomenon suggested that the critical degradation signal was present in the 12
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mutants. To further determine which PxxY motif was critical for WWP2-mediated
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degradation, we used the ΔPEFY, ΔPSGY, and ΔPEFYPSGY forms of Paip1 to analyze
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protein levels in the presence or absence of WWP2. Paip1-ΔPEFY and Paip1-ΔPSGY
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were both downregulated by WWP2. However, WWP2 did not downregulate
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Paip1-ΔPEFYPSGY (Fig. 4B). The two PxxY motifs and the PEFYPSGY sequence were
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necessary for WWP2 to recognize Paip1.
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We performed an ubiquitination assay to test whether or not the resistance of the
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mutant Paip1-ÄPEFYPSGY to WWP2-mediated degradation was caused by abrogated
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ubiquitination. WWP2 did not ubiquitylate the truncate (Fig. 4C). The PEFYPSGY
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sequence was critical in the catalytic activity of WWP2 during Paip1 ubiquitination and
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degradation.
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WWP2 negatively regulates Paip1-mediated translation enhancement. To gain
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insight into the functional relationship between WWP2 and Paip1, we initially determined
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the translational stimulatory activity via in vivo translation assays. Under the control of
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the Tet-off promoter expressing WWP2-WT or WWP2-C838 A, DNA vectors were
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transfected into HeLa cells along with constructs expressing Renilla luciferase and tTA.
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Each construct yielded comparable amounts of protein. Paip1 was absent in the extract
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expressing WWP2 (Fig. 5A). The relative induction of luciferase activity was determined
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by calculating the ratio of Renilla luciferase activity between induced (without Tet) and
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repressed (with Tet at 300 ng/ml) expressions of WWP2. Luciferase activity was lower
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with the overexpression of the wild-type WWP2 plasmid than with the expression of the 13
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vector alone (Fig. 5B lanes 1, 2, 3, 4, and 5). However, the overexpression of
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WWP2-C838 A did not affect in vivo translation (Fig. 5B lanes 6 and 7), thereby
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suggesting that the ligase activity of WWP2 was required. WWP2 overexpression did not
271
affect Renilla luciferase mRNA levels (Fig. 5C).
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To determine whether or not the translational suppression induced by WWP2
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depended on the presence of Paip1, we examined the effect of Paip1 silencing via siRNA
274
on WWP2 activity. The expressions of WWP2 and Paip1 were confirmed by Western
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blot analysis (Fig. 5D). No significant translational repression was observed when Paip1
276
was knocked down compared with the control (Fig. 5E). Renilla luciferase mRNA levels
277
were not affected by siRNA treatments (Fig. 5F). Therefore, WWP2 repressed mRNA
278
translation depending on the presence of Paip1. These data confirmed the importance of
279
the protein level regulation of Paip1 by WWP2. We demonstrated that WWP2 is a novel
280
regulator of translational initiation.
281 282
Discussion
283
Translation is important in the regulation of gene expression and is implicated in the
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control of cell growth, proliferation, and differentiation (32–34). In eukaryotes, initiation
285
is the rate-limiting step of translation under most circumstances; initiation is a major
286
target for regulation (33). Paip1 is a mammalian PABP that binds to eIF4A and eIF3 and
287
stimulates translational initiation. In the present study, we showed that Paip1 protein was
288
degraded by HECT ubiquitin ligase WWP2. The following findings from the present 14
289
study directly corroborate the use of Paip1 as a physiological substrate of WWP2. WWP2
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directly interacted with Paip1, and the interaction depended on the integrity of the WW
291
domain of WWP2. The loss of WWP2 impeded Paip1 turnover. WWP2 promoted Paip1
292
ubiquitination both in vivo and in a reconstituted in vitro system. The ubiquitin ligase
293
activity of WWP2 and the PxxY motif of Paip1 were critical for ubiquitination and
294
degradation. Previous studies have demonstrated the involvement of WWP2 in the
295
regulation of transcription, embryonic stem cell development, cellular transport, T-cell
296
activation processes, and tumorigenesis by targeting distinct substrates (35). In the
297
present study, we revealed that Paip1 was a novel substrate of WWP2. We also
298
emphasized the importance of WWP2 in the regulation of translation.
299
The involvement of deregulation of translational initiation in cancer development and
300
progression became the focus of various studies only recently. WWP2 mediates the
301
depletion of phosphatase and tensin homolog and consequently elevates
302
phosphoinositide-3-kinase–protein kinase B pathway activity (36). Such regulatory
303
activities may enhance eIF4E function and activate cap-dependent translation initiation,
304
leading to the selective increase of translation of key mRNAs; these mRNAs are involved
305
in tumor growth, angiogenesis, and cell survival (37). Thus, we hypothesized that
306
WWP2-mediated degradation of Paip1 selectively reduced the expression of numerous
307
potent growth and survival factors. The role of WWP2 in tumorigenesis, which depends
308
on WWP2’s regulation of translation, requires further investigation.
309
Circularization of the mRNA by bridging the mRNA 5’ and 3’ is an important step in 15
310
the process of translation initiation. In yeast, eIF4G interacts with PABP to contribute to
311
the circularization of the mRNA and mediation of the poly(A) tail-dependent translation
312
(11). In plants, PABP binds to eIF-iso4G and eIF4B, thereby increasing the RNA-binding
313
activity of PABP (13). However, mammalian cells possess dual systems, namely,
314
PABP–Paip1 and PABP–eIF4G. These systems regulate the formation of mRNP. The
315
presence of Paip1 in animal cells reflects evolutionary advancement and allows higher
316
eukaryotes to link PABP to eIF4A function. Information is lacking on whether the
317
degradation of Paip1 by WWP2 reduces the rate of the circular mRNA conformation and
318
inhibits mRNA translation. Paip1’s sequence and function are similar to those of the
319
eIF4G protein. WWP2 may also regulate the protein level of eIF4G in human cells, but
320
the relationship between WWP2 and eIF4G requires further study.
321
The stimulation of translation by Paip1 in vivo decreased upon the deletion of the
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N-terminal sequence containing PABP and the eIF3 binding domain known as PAM2
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(22). In the present study, PAM2 was critical for the binding of PABP to WWP2.
324
Paip1-interacting proteins can potentially compete for Paip1 binding via the PAM2
325
domain, thereby regulating translation. Further studies are required to determine the
326
presence of an overlap among the binding sites of WWP2, eIF3, and PABP in Paip1.
327
WWP2 was identified as a typical ubiquitin ligase for Paip1. We showed that the
328
overexpression of WWP2 significantly decreased Paip1 protein level, and such decrease
329
suppressed translation. We also discovered that the WW domain of WWP2 interacted
330
with Paip1 fragments, which contained two PxxY motifs. The degradation and 16
331
ubiquitination of Paip1 by WWP2 was transiently abolished by the deletion of the two
332
PxxY motifs. WWP2 is an important translational suppressor that participates in Paip1
333
degradation. The function of WWP2 in the regulation of translation was clarified for the
334
first time. The mechanisms underlying WWP2’s regulation of Paip1 should be studied
335
further to reveal the details of WWP2’s function.
336 337
ACKNOWLEDGMENTS
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This work was supported by grants from the Nature Science of Shandong Province,
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Q2008C08. The funders had no role in the study design, data collection, data analysis,
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decision to publish, or preparation of the manuscript.
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REFERENCES
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1.
Parsyan A, Hernandez G, Meterissian S. 2012. Translation initiation in colorectal cancer. Cancer Metastasis Rev 31:387-395.
2.
Jia Y, Polunovsky V, Bitterman PB, Wagner CR. 2012. Cap-dependent translation initiation factor eIF4E: an emerging anticancer drug target. Med Res Rev 32:786-814.
3.
Graff JR, Konicek BW, Carter JH, Marcusson EG. 2008. Targeting the eukaryotic translation initiation factor 4E for cancer therapy. Cancer research 68:631-634.
4.
Cheung YN, Maag D, Mitchell SF, Fekete CA, Algire MA, Takacs JE, Shirokikh N, Pestova T, Lorsch JR, Hinnebusch AG. 2007. Dissociation of eIF1 from the 40S ribosomal subunit is a key step in start codon selection in vivo. Genes Dev 21:1217-1230.
5.
Chiu WL, Wagner S, Herrmannova A, Burela L, Zhang F, Saini AK, Valasek L, Hinnebusch AG. 2010. The C-terminal region of eukaryotic translation initiation factor 3a (eIF3a) promotes mRNA recruitment, scanning, and, together with eIF3j and the eIF3b RNA recognition motif, selection of AUG start codons. Molecular and cellular biology 30:4415-4434.
6.
Pisarev AV, Unbehaun A, Hellen CU, Pestova TV. 2007. Assembly and analysis of eukaryotic translation initiation complexes. Methods Enzymol 430:147-177.
7.
Lorsch JR, Dever TE. 2010. Molecular view of 43 S complex formation and start site selection in eukaryotic translation initiation. THE JOURNAL OF BIOLOGICAL CHEMISTRY 285:21203-21207. 17
360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400
8.
Preiss T, M WH. 2003. Starting the protein synthesis machine: eukaryotic translation initiation. Bioessays 25:1201-1211.
9.
Prevot D, Darlix JL, Ohlmann T. 2003. Conducting the initiation of protein synthesis: the role of eIF4G. Biol Cell 95:141-156.
10.
Kessler SH, Sachs AB. 1998. RNA recognition motif 2 of yeast Pab1p is required for its functional interaction with eukaryotic translation initiation factor 4G. Molecular and cellular biology 18:51-57.
11.
Tarun SZ, Jr., Sachs AB. 1996. Association of the yeast poly(A) tail binding protein with translation initiation factor eIF-4G. The EMBO Journal 15:7168-7177.
12.
Tarun SZ, Jr., Wells SE, Deardorff JA, Sachs AB. 1997. Translation initiation factor eIF4G mediates in vitro poly(A) tail-dependent translation. Proceedings of the National Academy of Sciences of the United States of America 94:9046-9051.
13.
Le H, Tanguay RL, Balasta ML, Wei CC, Browning KS, Metz AM, Goss DJ, Gallie DR. 1997. Translation initiation factors eIF-iso4G and eIF-4B interact with the poly(A)-binding protein and increase its RNA binding activity. THE JOURNAL OF BIOLOGICAL CHEMISTRY 272:16247-16255.
14.
Khaleghpour K, Kahvejian A, De Crescenzo G, Roy G, Svitkin YV, Imataka H, O'Connor-McCourt M, Sonenberg N. 2001. Dual interactions of the translational repressor Paip2 with poly(A) binding protein. Molecular and cellular biology 21:5200-5213.
15.
Roy G, De Crescenzo G, Khaleghpour K, Kahvejian A, O'Connor-McCourt M, Sonenberg N. 2002. Paip1 interacts with poly(A) binding protein through two independent binding motifs. Mol Cell Biol 22:3769-3782.
16.
Imataka H, Olsen HS, Sonenberg N. 1997. A new translational regulator with homology to eukaryotic translation initiation factor 4G. The EMBO Journal 16:817-825.
17.
Levy-Strumpf N, Deiss LP, Berissi H, Kimchi A. 1997. DAP-5, a novel homolog of eukaryotic translation initiation factor 4G isolated as a putative modulator of gamma interferon-induced programmed cell death. Molecular and cellular biology 17:1615-1625.
18.
Yamanaka S, Poksay KS, Arnold KS, Innerarity TL. 1997. A novel translational repressor mRNA is edited extensively in livers containing tumors caused by the transgene expression of the apoB mRNA-editing enzyme. Genes Dev 11:321-333.
19.
Morino S, Imataka H, Svitkin YV, Pestova TV, Sonenberg N. 2000. Eukaryotic translation initiation factor 4E (eIF4E) binding site and the middle one-third of eIF4GI constitute the core domain for cap-dependent translation, and the C-terminal one-third functions as a modulatory region. Molecular and cellular biology 20:468-477.
20.
Imataka H, Sonenberg N. 1997. Human eukaryotic translation initiation factor 4G (eIF4G) possesses two separate and independent binding sites for eIF4A. Molecular and cellular biology 17:6940-6947.
21.
Craig AW, Haghighat A, Yu AT, Sonenberg N. 1998. Interaction of polyadenylate-binding protein with the eIF4G homologue PAIP enhances translation. Nature 392:520-523.
22.
Martineau Y, Derry MC, Wang X, Yanagiya A, Berlanga JJ, Shyu AB, Imataka H, Gehring K, Sonenberg N. 2008. Poly(A)-binding protein-interacting protein 1 binds to eukaryotic translation initiation factor 3 to stimulate translation. Mol Cell Biol 28:6658-6667. 18
401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437
23.
Martineau Y, Wang X, Alain T, Petroulakis E, Shahbazian D, Fabre B, Bousquet-Dubouch MP, Monsarrat B, Pyronnet S, Sonenberg N. 2014. Control of Paip1-eukayrotic translation initiation factor 3 interaction by amino acids through S6 kinase. Mol Cell Biol 34:1046-1053.
24.
Zou W, Chen X, Shim JH, Huang Z, Brady N, Hu D, Drapp R, Sigrist K, Glimcher LH, Jones D. 2011. The E3 ubiquitin ligase Wwp2 regulates craniofacial development through mono-ubiquitylation of Goosecoid. Nature Cell Biology 13:59-65.
25.
Nakamura Y, Yamamoto K, He X, Otsuki B, Kim Y, Murao H, Soeda T, Tsumaki N, Deng JM, Zhang Z, Behringer RR, Crombrugghe B, Postlethwait JH, Warman ML, Nakamura T, Akiyama H. 2011. Wwp2 is essential for palatogenesis mediated by the interaction between Sox9 and mediator subunit 25. Nature Communications 2:251.
26.
Maddika S, Kavela S, Rani N, Palicharla VR, Pokorny JL, Sarkaria JN, Chen J. 2011. WWP2 is an E3 ubiquitin ligase for PTEN. Nature Cell Biology 13:728-733.
27.
Soond SM, Chantry A. 2011. Selective targeting of activating and inhibitory Smads by distinct WWP2 ubiquitin ligase isoforms differentially modulates TGFbeta signalling and EMT. Oncogene 30:2451-2462.
28.
Gossen M, Bujard H. 1992. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proceedings of the National Academy of Sciences of the United States of America 89:5547-5551.
29.
Bernassola F, Karin M, Ciechanover A, Melino G. 2008. The HECT family of E3 ubiquitin ligases: multiple players in cancer development. Cancer Cell 14:10-21.
30.
Albrecht M, Lengauer T. 2004. Survey on the PABC recognition motif PAM2. Biochem Biophys Res Commun 316:129-138.
31.
Ingham RJ, Gish G, Pawson T. 2004. The Nedd4 family of E3 ubiquitin ligases: functional diversity within a common modular architecture. Oncogene 23:1972-1984.
32. 33.
Conlon I, Raff M. 1999. Size control in animal development. Cell 96:235-244. Gingras AC, Raught B, Sonenberg N. 1999. eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation. Annu Rev Biochem 68:913-963.
34.
Miron M, Verdu J, Lachance PE, Birnbaum MJ, Lasko PF, Sonenberg N. 2001. The translational inhibitor 4E-BP is an effector of PI(3)K/Akt signalling and cell growth in Drosophila. Nature Cell Biology 3:596-601.
35.
Chen A, Gao B, Zhang J, McEwen T, Ye SQ, Zhang D, Fang D. 2009. The HECT-type E3 ubiquitin ligase AIP2 inhibits activation-induced T-cell death by catalyzing EGR2 ubiquitination. Molecular and cellular biology 29:5348-5356.
36.
Chantry A. 2011. WWP2 ubiquitin ligase and its isoforms: new biological insight and promising disease targets. Cell cycle 10:2437-2439.
37.
Graff JR, Konicek BW, Carter JH, Marcusson EG. 2008. Targeting the eukaryotic translation initiation factor 4E for cancer therapy. Cancer Res 68:631-634.
438 439 19
440
FIGURE LEGENDS
441
Fig. 1. WWP2 negatively regulates Paip1 protein level. (A) MG132 treatment upregulates
442
Paip1 protein level. HEK293T cells were treated with MG132 (20 µM) for 8 h before
443
harvest. Endogenous Paip1, PABP, and Paip2 levels were analyzed by immunoblotting.
444
GAPDH was used as an internal control. (B) Schematic of Paip1 protein. The positions of
445
PxxY motifs are labeled. (C) WWP2 specifically reduces Paip1 protein amount. The
446
indicated Nedd4 family of E3 ligases was co-transfected with Paip1 into HEK293T cells,
447
and cell lysates were analyzed by immunoblotting. (D) WWP2 downregulates Paip1 in a
448
proteasome-dependent manner. Increasing concentrations of Flag-WWP2 plasmids were
449
co-transfected into HEK293T cells with Myc-Paip1. Cells were treated with MG132
450
(20 μM) and lactacystin (30 μM) or dimethyl sulfoxide (DMSO) for 16 h. The cell lysates
451
were analyzed by immunoblotting. (E) WWP2 decreases the Paip1 protein level in a
452
dose-dependent manner. Cells were transfected with increasing amounts of WWP2-WT
453
or the catalytic mutant form WWP2-C838A. Subsequently, Paip1 level was detected. (F)
454
WWP2 can not affect the mRNA level of Paip1. Paip1 mRNA prepared from the
455
transfected HEK293T cells was analyzed by real-time PCR assay. Data are presented as
456
mean ± S.D. (n = 3). (G) WWP2 depletion increases the endogenous Paip1 protein level.
457
HEK293T and HeLa cells were transfected with non-targeted control or with
458
WWP2-specific siRNA. The endogenous WWP2 and Paip1 levels were analyzed by
459
Western blot. (H) WWP2 depletion can not affect the Paip1 mRNA level. Paip1 mRNA
460
prepared from the transfected HEK293T and HeLa cells was analyzed by real-time PCR 20
461
assay. Data are presented as mean ± S. D. (n = 3). (I) WWP2 can not affect the
462
endogenous Paip2 and PABP protein levels. Cells were transfected with the WWP2-WT
463
or the catalytic mutant form WWP2-C838A. Paip2 and PABP protein levels were
464
detected by immunoblotting (top). HEK293T cells were transfected with non-targeted
465
control or WWP2–specific siRNA. The endogenous WWP2 and PABP protein levels
466
were analyzed by immunoblotting (bottom). (J) Overexpression of Paip2 can not affect
467
WWP2-mediated degradation of Paip1. HEK293T cells were transfected with the vector
468
or with Paip2. At 24 h after transfection, cells were transfected with WWP2 and analyzed
469
by immunoblotting.
470
Fig. 2. WWP2 promotes the ubiquitination and degradation of Paip1. (A) WWP2 reduces
471
the half-life of endogenous Paip1 protein. HEK293T cells were transfected with control
472
plasmid or Flag-WWP2-WT or the catalytic mutant form Flag-WWP2-C838 A, and cells
473
were treated with the protein synthesis inhibitor CHX (10 µg/ml) for the indicated
474
durations before harvest. Paip1 protein expression was analyzed. Quantitative analysis
475
was performed by measuring integrated optical density using the program Gel-Pro
476
analyzer. Data are presented as mean ± S. D. (n = 3). (B) Depletion of WWP2 prolonges
477
the half-life of endogenous Paip1 protein. HEK293T cells were transfected with control
478
siRNA or WWP2 siRNA and treated with CHX. Paip1 protein expression was
479
subsequently analyzed. Quantitative analysis was performed by measuring integrated
480
optical density using the program Gel-Pro analyzer. Data are presented as mean ± S. D. (n
481
= 3). (C) WWP2 catalyzes the ubiquitination of Paip1 in vitro. A mixture comprising 21
482
purified HA-ubiquitin, E1, E2 (UbcH5c), bacteria-expressed and purified
483
His-WWP2-WT or C838A, and GST-Paip1 or GST was used for in vitro ubiquitination
484
assays and subsequent immunoblotting with anti-HA. (D) WWP2 enhances the
485
ubiquitination of Paip1 in vivo. HEK293T cells were transfected with HA-Ub,
486
Myc-Paip1, control vector, or Flag-WWP2, and treated with MG132 as indicated.
487
Ubiquitinated Paip1 was immunoprecipitated by anti-Myc antibody and analyzed by
488
immunoblotting. (E) WW and HECT domains of WWP2 were involved in the
489
ubiquitination of Paip1. HA-Ub, Myc-Paip1, and WWP2 constructs were co-transfected
490
into HEK293T cells and treated with MG132. Ubiquitinated Paip1 was
491
immunoprecipitated with anti-HA antibody and analyzed by immunoblotting.
492
Fig. 3. Mapping of the interaction region between the truncated domains of WWP2 and
493
Paip1. (A) WWP2 was colocalized with Paip1 within the cytoplasm. Myc-Paip1 and
494
Flag-WWP2 were co-transfected into HEK293T cells, and cells were stained 24 h later
495
using mouse anti-Flag and rabbit anti-Myc antibodies for visualization by confocal
496
microscopy. (B) Paip1 was co-immunoprecipitated with wild type and catalytic mutant
497
forms of WWP2. Wild type and catalytic mutant forms of WWP2 and Myc-Paip1 were
498
transfected into HEK293T cells. Cell lysates were immunoprecipitated with anti-Flag
499
antibody. Lysates and immunoprecipitates were analyzed by immunoblotting. (C)
500
Immunoprecipitation analysis of WWP2 and Paip1 in vivo. HeLa cells were transfected
501
with negative control siRNA or siRNA targeted Paip1, and cells were harvested after 36 h.
502
The extracts were immunoprecipitated with anti-WWP2 antibody and subsequently 22
503
analyzed by immunoblotting using anti-WWP2, anti-Paip1, anti-eIF3a, and anti-PABP
504
antibodies. Pre-immune IgG was used as the control. (D) Direct interaction between the
505
WW domain of WWP2 and Paip1. GST pull-downs were conducted with GST or
506
GST-WWP2 fragments along with purified His-Paip1. Input and pull-down samples were
507
subjected to immunoblotting with anti-GST and anti-His antibodies. Input represented 10%
508
of the sample used for the pull-down. (E) PAM2 motif of Paip1 showed direct binding to
509
WWP2. GST pull-downs were performed with GST or GST-Paip1 fragments along with
510
purified His-WWP2. Input and pull-down samples were subjected to immunoblotting
511
with anti-GST and anti-His antibodies. Input represented 10% of the sample used for
512
pull-down. (F) WWP2 can not affect the interaction between Paip1 and PABP. HEK293T
513
cells were transfected with Flag-WWP2-WT or the catalytic mutant form
514
Flag-WWP2-C838A. Cell lysates were immunoprecipitated with anti-Paip1 antibody.
515
Both the lysate and immunoprecipitates were analyzed by immunoblotting (left).
516
HEK293T cells were transfected with control siRNA or WWP2 siRNA. Cell lysates were
517
immunoprecipitated with anti-Paip1 antibody. Lysates and immunoprecipitates were
518
analyzed by immunoblotting (right).
519
Fig. 4. PEFYPSGY sequence is sufficient for the ubiquitination and degradation of Paip1.
520
(A) HEK293T cells were transfected with plasmids expressing either full-length Paip1 or
521
mutants of Paip1 with or without a plasmid encoding Flag-WWP2-WT. (B) PEFYPSGY
522
sequence maintains the WWP2-mediated degradation of Paip1. HEK293T cells were
523
transfected with plasmids expressing Paip1-ΔPEFY, Paip1-ΔPSGY, or 23
524
Paip1-ΔPEFYPSGY with or without Flag-WWP2. The cell lysates were analyzed by
525
immunoblotting. (C) PEFYPSGY sequence attenuated WWP2-mediated ubiquitination of
526
Paip1. HEK293T cells were co-transfected with Myc-Paip1 deletions and HA-Ub with or
527
without Flag-WWP2. Ubiquitinated Paip1 was immunoprecipitated with an anti-Myc
528
antibody and protein A/G-agarose beads under denaturing conditions to eliminate any
529
WWP2-associated protein by non-covalent bonding.
530
Fig. 5. Overexpression of WWP2 represses translation via the Paip1 protein. (A) Cells
531
were transfected with the indicated pTet-HA-WWP2-WT or catalytic mutant form
532
pTet-HA-WWP2-C838A plasmids along with constructs expressing Renilla luciferase
533
and tTA. Cells were cultured in a medium containing 0 or 300 ng/ml of Tet. Cells were
534
harvested and subjected to Western blot analysis with the indicated antibodies. (B)
535
Renilla luciferase activity was quantified in HeLa cell extracts harvested in (A) and
536
normalized on the total protein level. Relative induction of the luciferase reporter was
537
determined by calculating the ratio of Renilla luciferase activity between induced
538
(without Tet) and repressed (with 300 ng/ml Tet) expression of the indicated HA-WWP2.
539
Error bars denote the standard error of the mean for the three independent experiments.
540
(C) Quantitative real-time PCR of Renilla luciferase mRNA obtained from total RNA of
541
duplicate HeLa cells harvested in (A). Data are presented as mean ± S. D. (n = 3). (D)
542
HeLa cells were transfected with the indicated siRNA sequences. At 24 h after siRNA
543
transfection, cells were transfected as described in (A). Cells were subsequently placed in
544
a medium containing 0 or 300 ng/ml of Tet. Cells were harvested and subjected to 24
545
Western blot analysis with the indicated antibodies. (E) Renilla luciferase activity was
546
quantified in HeLa cell extracts harvested in (D) and normalized on the total protein level.
547
Relative induction of the luciferase reporter was determined by calculating the ratio of
548
Renilla luciferase activity between induced (without Tet) and repressed (with 300 ng/ml
549
Tet) expression of the indicated HA-WWP2. Error bars denote the standard error of the
550
mean for three independent experiments. (F) Quantitative RT–PCR of Renilla luciferase
551
mRNA obtained from the total RNA of the duplicate HeLa cells harvested in (D). Data
552
are presented as mean ± S. D. (n = 3).
25
1
Paip1, an effective stimulator of translation initation, is targeted by
2
WWP2 for ubiquitination and degradation
3 4
Yanrong Lv1, Kai Zhang1, Haidong Gao1*
5 6
Department of Breast Surgery, QiLu Hospital of Shandong University, Jinan, Shandong,
7
China1
8 9 10
Running title: WWP2 ubiquitylates and degrades Paip 1
11 12 13 14 15
* Corresponding author. Haidong Gao, Department of Breast Surgery, QiLu Hospital of Shandong University No. 107, Wen hua Xi Road, Jinan, China, Tel: +86 15806661668, E-mail: [email protected] 1
16
ABSTRACT
17
Poly(A)-binding protein-interacting protein 1 (Paip1) stimulates translational initiation by
18
inducing the circularization of mRNA. However, the mechanisms underlying Paip1
19
regulation, particularly its protein stability, are still unclear. Here we show that the E6AP
20
carboxyl terminus (HECT)-type ubiquitin ligase WW domain-containing protein 2
21
(WWP2), a homolog of HECT-type ubiquitin ligase WWP1, interacts with and targets
22
Paip1 for ubiquitination and proteasomal degradation. Mapping of the region, including
23
the WW domain of WWP2, revealed the interaction between WWP2 and the
24
PABP-binding motif 2 (PAM2) of Paip1. The two consecutive PxxY motifs in PAM2 are
25
required for WWP2-mediated ubiquitination and degradation. Furthermore, ectopic
26
expression of WWP2 decreases translational stimulatory activity with the degradation of
27
Paip1. We therefore provide the evidence that the stability of Paip1 can be regulated by
28
ubiquitin-mediated degradation; thus, highlighting the importance of WWP2 as a
29
suppressor of translation.
30 31
INTRODUCTION
32
Regulation of gene expression occurs in eukaryotes during messenger RNA (mRNA)
33
translation, specifically at the initiation of translation. Deregulation at this step of the
34
translation process leads to abnormal gene expression, which in turn alters cell growth
35
and possibly leads to cancer development (1–3). Translational initiation comprises a
36
series of discrete steps and starts with the dissociation of 80S ribosomes into subunits. 2
37
The eukaryotic translational initiation factors (eIFs) 1, 1A, and 5, and the eIF3 complex
38
promote the binding of the eIF2-GTP-Met-tRNAi ternary complex to the 40S subunit,
39
thereby forming a 43S pre-initiation complex (PIC) (4–7). The 43S PIC is loaded onto the
40
mRNA near the 5’-7-methylguanosine cap by numerous factors, including eIF3,
41
poly(A)-binding protein (PABP), eIF4B, and eIF4F complex. The eIF4F complex
42
comprises three subunits, namely, a cap-binding protein (eIF4E), an RNA helicase
43
(eIF4A), and a scaffold protein eIF4G (8,9). eIF4G harbors the binding domains for
44
PABP, eIF4E, eIF4A, and eIF3 in mammals. The binding domains for eIF4E and PABP
45
in eIF4G enable the assembly of a stable, circular messenger ribonucleoprotein (mRNP)
46
by eIF4G; eIF4G–eIF3 interaction generates a protein bridge between the mRNPs
47
(10–13).
48
PABP-interacting protein 1 (Paip1) is a PABP-binding protein that contains two
49
distinct PABP-binding motifs (PAMs). PAM1 binds to RNA recognition motif 2 in the N
50
terminus of PABP; PAM2, which is a conserved region comprising approximately 15
51
amino acids, binds to the PABC domain of PABP (14, 15). Paip1 shows 39% similarity
52
to eIF4G and the eIF4G-related protein, p97/DAP5/NAT1 (16–18). A specific portion
53
that is present in both Paip1 and eIF4G has one of two known eIF4A binding regions and
54
an eIF3 binding site (19, 20). The abovementioned findings indicate that Paip1
55
co-immunoprecipitates with eIF4A and eIF3 (21, 22). The presence of Paip1 in animal
56
cells may indicate the involvement of a mechanism that links PABP to eIF4A, thereby
57
causing the circularization of mRNA (21). Data from previous studies suggest that the 3
58
interaction of Paip1 with eIF3 stabilizes circular mRNP conformation, which is formed
59
by eIF4G–PABP interaction (22). eIF3 is reportedly phosphorylated by S6K1/2, which
60
stimulates the Paip1–eIF3 interaction and the initiation of translation (23). Paip1 is an
61
important positive effector of translational initiation, but information is lacking on the
62
mechanism underlying Paip1 regulation.
63
In the present study, we demonstrated the critical function of an E6AP carboxyl
64
terminus (HECT)-domain comprising an E3 ubiquitin ligase WW domain-containing
65
protein 2 (WWP2), which is also known as atrophin-1 interacting protein 2 (AIP2), in the
66
regulation of Paip1 protein stability. WWP2 is homologous to the HECT domain-type
67
ubiquitin-protein ligase and participates in the regulation of craniofacial development and
68
chondrogenesis (24, 25). WWP2 also participates in the maintenance of key oncogenic
69
signaling pathways, which are linked to cancer cell growth, survival, and tumor spread
70
(26, 27). Here we showed that WWP2 interacted with the PAM2 motif of Paip1 via the
71
WW domain. Further investigation revealed that WWP2 targeted Paip1 for ubiquitination
72
and degradation via the “PEFYPSGY” sequence in the PAM2 motif. Importantly, WWP2
73
was found to participate in translational initiation by regulating Paip1 protein level.
74
MATERIALS AND METHODS
75
Cell Culture and Transfection. HEK293T and HeLa cells were cultured in Dulbecco’s
76
modified Eagle’s medium supplemented with 10% fetal bovine serum and 5 U/ml
77
penicillin-streptomycin (Gibco, USA) in 5% CO2. Cells were transfected by
78
Lipofectamine 2000 following the manufacturer’s protocol (Invitrogen, USA). 4
79
Antibodies and Reagents. The proteasomal inhibitors MG132 and lactacystin were
80
purchased from Sigma-Aldrich, USA. The WWP2 and Paip1 antibodies were purchased
81
from Abcam, UK. Anti-glyceraldehyde 3-phosphate dehydrogenase (Anti-GAPDH) and
82
secondary antibodies were purchased from Santa Cruz Biotechnology, Inc., USA.
83
Anti-hemagglutinin (Anti-HA) was obtained from Roche Applied Science, Germany, and
84
anti-Myc and anti-Flag antibodies were from MBL.
85
Immunoprecipitation and Immunoblotting. For general cell lysis, transfected cells
86
were harvested and lysed in HEPES lysis buffer (containing 20 mM HEPES, pH 7.2,
87
50 mM NaCl, 0.5% Triton X-100, 1 mM NaF, and 1 mM dithiothreitol) and boiled with
88
2× SDS–PAGE loading buffer. For immunoprecipitation, cell lysates were prepared in
89
500 ml HEPES buffer supplemented with a protease inhibitor mixture (Roche Applied
90
Science). Immunoprecipitation was performed by primary antibody incubation for 3 h
91
followed by overnight incubation with Protein A/G Sepharose beads (Santa Cruz). The
92
beads were washed with HEPES buffer thrice and examined by immunoblotting.
93
Immunofluorescence. For subcellular localization analyses, cells were fixed with 4%
94
paraformaldehyde and permeabilized in 0.2% Triton X-100 (phosphate-buffered saline).
95
Proteins were stained using the indicated antibodies and detected with a TRITC- or
96
FITC-conjugated secondary antibody. The nuclei were stained with 4′,6′
97
-diamidino-2-phenylindole hydrochloride (DAPI; Sigma), and images were visualized
98
with a Zeiss LSM 510 Meta inverted confocal microscope.
99
RNA Interference. The WWP2 siRNA-1 (5’- CACCTACTTTCGCTTTATA5
100
3’), siRNA-2 (5’- GGAGTACGTGCGCAACTAT-3’), and non-targeting siRNAs
101
(5’-UUCUCCGAACGUGUCACGU-3’) were synthesized by Shanghai GenePharm. All
102
siRNAs were transfected into the cells according to the manufacturer’s protocol.
103
Determination of turnover of Paip1. HEK293T cells were transfected with Myc-Paip1
104
with Flag-WWP2, Flag-WWP2-C838A, or empty vector (or interfering RNA). The cells
105
were treated with cycloheximide (CHX, 15 mg/ml, Sigma) at 36 h after transfection and
106
harvested at the indicated time. The protein level was measured by Western blot using the
107
indicated antibodies.
108
Ubiquitination assays. Cells were transfected with Myc-Paip1 and Flag-WWP2 or its
109
mutants to investigate Paip1 ubiquitination. Subsequently, cells were treated with MG132
110
(20 ìM) for 8 h before harvest. Cell lysis solution was prepared in modified RIPA lysis
111
buffer containing 10 mM Tris–HCl, pH 7.5, 150 mM NaCl, 5 mM
112
ethylenediaminetetraacetic acid (EDTA), 1% NP-40, 1% sodium deoxycholate, 0.025%
113
sodium dodecyl sulfate, and protease inhibitors. Immunoprecipitation was conducted
114
using the indicated antibody. Subsequently, immunoblot analysis was performed. For in
115
vitro ubiquitination assay, His-WWP2 and glutathione s-transferase (GST)-Paip1 were
116
expressed in Escherichia coli and were purified with Ni-nitrilotriacetate-agarose (Qiagen,
117
Netherlands) and glutathione-Sepharose 4B beads (Amersham, UK), respectively. The
118
assays were conducted in 30 il ubiquitination assay buffer (containing 50 mM Tris, pH
119
8.0, 50 mM NaCl, 1 mM dithiothreitol, 5 mM MgCl2, and 3 mM ATP) with 0.7 ìg of E1,
120
1 ìg of UbcH5c (E2), 15 ìg of HA-ubiquitin (Boston Biochem, MA, USA), 0.7 ìg of 6
121
His-WWP2 (wild-type or C838A mutant), and 1.5 ìg GST–Paip1. Samples were
122
incubated at 30 °C for 2 h. Reactions were terminated using the sample buffer.
123
Real-time reverse transcription–polymerase chain reaction (RT–PCR). Total RNA
124
was isolated from the cells using TRIZOL (Invitrogen) and reverse transcribed using 1 ìg
125
of total RNA with an oligo(dT) primer. The following primers were used for real-time
126
PCR: human GAPDH forward,
127
5’-GGGAAGGTGAAGGTCGGAGT-3’; GAPDH reverse, 5’-
128
TTGAGGTCAATGAAGGGGTCA-3’; human WWP2 forward,
129
5’- CGCAACTATGAGCAGTGGCA-3’; human WWP2 reverse,
130
5’- GGTCGTGCGAGTGTTATGGT-3’; human Paip1 forward, 5’-
131
GGAGAACTGGAAAGCCGAGGGTA-3’; and human Paip1 reverse,
132
5’- GTGTAACTGGAAGAATAACCTGAAGGG-3’; Renilla forward
133
5’-CAGTGGTGGGCCAGATGTAAACAA-3’; and Renilla reverse
134
5’-TAATACACCGCGCTACTGGCTCAA-3’.
135
Translation assays. HeLa cells were grown in 60 mm dishes and co-transfected with
136
Myc-Paip1, pTet-HA-WWP2 (WT or CA) or control vector pUHD-15-1 expressing the
137
Tet-controlled transactivator (tTA) (28), and pRL-CMV (Promega, USA). pRL-CMV is a
138
Renilla luciferase reporter construct. The medium was replaced with Tet at a final
139
concentration of 0 or 300 ng/ml after 4 h. Cells were harvested 48 h after transfection,
140
and extracts were used to quantify Renilla luciferase by a dual-luciferase reporter assay
141
system (Promega). Protein concentration was determined using Bio-Rad protein assay 7
142
reagent, and Renilla luciferase activity was corrected according to the protein
143
concentration. The relative induction for each construct was determined by calculating
144
the ratio of Renilla luciferase activity between the induced condition (no Tet) and the
145
repressed condition (with 300 ng/ml Tet). Extracts were subjected to SDS–PAGE and
146
Western blot analysis.
147
Results
148
WWP2 negatively regulates the protein level of Paip1 in a proteasome-dependent
149
manner. To explored the mechanism underlying Paip1 regulation, we first investigated
150
whether or not the Paip1 protein was regulated through the proteasome pathway, which is
151
the most critical pathway in the regulation of cellular protein stability and quality in
152
eukaryotes. The level of endogenous Paip1 protein increased significantly after MG132
153
treatment in both HEK293T and HeLa cells (Fig. 1A), thereby suggesting that Paip1
154
protein regulation depended on the proteasome system. We also examined the effect of
155
MG132 treatment on the levels of other related translation factors, such as PABP and
156
Paip2. The proteasome inhibitors failed to affect the PABP and Paip2 protein levels (Fig.
157
1A). The ubiquitin ligases controlled ubiquitination and determined the specificity of
158
substrate recognition during ubiquitin-mediated proteasomal degradation. So we
159
determined which ubiquitin ligase downregulated Paip1 protein stability. Sequence
160
analysis showed that Paip1 comprised three putative PxxY-type PY motifs (Fig. 1B).
161
These motifs are recognized by the Nedd4 family of ubiquitin ligases. Among the nine
162
members of the Nedd4 ligases, only WWP2 specifically downregulated Paip1 protein 8
163
levels (Fig. 1C). The downregulation of Paip1 by WWP2 was blocked by proteasome
164
inhibitors MG132 and lactacystin (Fig. 1D). Thus, we hypothesized that WWP2 functions
165
as an ubiquitin ligase in Paip1 degradation.
166
To test our hypothesis, we transiently transfected HEK293T cells with a constant
167
amount of Paip1 and an increasing amount of wild-type WWP2 or ligase-inactive
168
WWP2-C838A expression vectors. The Paip1 protein level progressively decreased with
169
increasing WWP2-WT expression, but this phenomenon was not observed with increasing
170
WWP2-C838A mutant expression (Figs. 1E and 1F). No change in the Paip1 mRNA level
171
was observed. Thus, WWP2 could mediate Paip1 destruction depending on its ubiquitin
172
ligase activity.
173
To verify whether endogenous WWP2 regulates Paip1 level, WWP2 was depleted
174
using two independent siRNAs. The depletion of endogenous WWP2 in HEK293T and
175
HeLa cells significantly increased Paip1 protein level (Fig. 1G). However, the mRNA
176
level was not affected by the abovementioned changes (Fig. 1H). WWP2 can function as
177
an ubiquitin ligase to maintain proper Paip1 protein levels in human cells.
178
Paip1 and Paip2 share similar domains and compete for PABP binding. According
179
to previous studies, another HECT-type ubiquitin ligase, namely, EDD, targets Paip2 for
180
degradation during PABP depletion. We examined the effect of WWP2 overexpression
181
on Paip2 and found that WWP2 did not affect Paip2 protein levels (Fig. 1I). Furthermore,
182
overexpression of Paip2 did not affect WWP2-mediated Paip1 degradation (Fig. 1J).
183
Paip1 and Paip2 did not compete for WWP2 binding, and the degradation of the two 9
184
proteins required different ubiquitin ligases. Moreover, the Paip1 binding protein, PABP,
185
was not regulated upon WWP2 silencing and overexpression (Fig. 1I).
186
WWP2 promotes the degradation and ubiquitination of Paip1. To assessed
187
whether the reduction in the protein level of Paip1, which was induced by WWP2
188
overexpression, was due to increasing Paip1 degradation. We analyzed the steady-state
189
levels of Paip1 by applying CHX, an inhibitor of protein synthesis. The half-life of
190
exogenously expressed Paip1 was greatly reduced by the expression of WWP2-WT, but
191
not by the expression of Paip1-C838 A (Fig. 2A). However, the depletion of WWP2
192
prolonged the half-life of endogenous Paip1 (Fig. 2B). Thus WWP2 promoted the
193
degradation of the Paip1 protein.
194
We determined whether WWP2 functioned as an ubiquitin ligase and if WWP2
195
directly promoted the ubiquitination of Paip1. An in vitro ubiquitination assay was
196
initially conducted using purified E1 and UbcH5c, bacteria-expressed His-WWP2-WT or
197
His-WWP2-C838A, and GST-Paip1 or GST (control). Bacteria-expressed and purified
198
WWP2-WT promoted the ubiquitination of Paip1, but such effect on Paip1 was not
199
induced by the WWP2-C838A (Fig. 2C). Overexpressed WWP2 enhanced the
200
ubiquitination of Paip1 in the presence of MG132 in HEK293T cells (Fig. 2D). The
201
required WW and HECT domains were sufficient for WWP2 to promote the degradation
202
of Paip1 (Fig. 2E). These data suggested that WWP2 functioned as a biologically active
203
ubiquitin ligase and induced the ubiquitination and degradation of Paip1.
204
The WW domain of WWP2 is required for binding to the PAM2 motif of Paip1. 10
205
Proteins should at least partly colocalize to be functionally linked. Indirect
206
immunofluorescence assays revealed that WWP2 and Paip1 colocalized predominantly in
207
the cytoplasm (Fig. 3A). We further confirmed the interaction of WWP2 with Paip1 in
208
mammalian cells. A co-immunoprecipitation assay revealed the association between
209
Paip1 and WWP2-WT or C838A mutant (Fig. 3B). Thus, ubiquitin ligase activity was not
210
required for the interaction. In addition, endogenous Paip1 and its binding partners were
211
co-immunoprecipitated with WWP2, but not with the control IgG from HeLa cells
212
(Fig. 3C left panel). Given the interactions between WWP2 and eIF3a, PABP disappeared
213
in a Paip1 depletion cell extract (Fig. 3C right panel), thereby suggesting that eIF3a and
214
PABP bonded to WWP2 depending on the presence of Paip1.
215
The direct association between WWP2 and Paip1 was confirmed by GST pull-down
216
assay. WWP2 protein comprised three domains, namely, C2, WW (including four tandem
217
WW domains), and HECT. The results of the GST pull-down assays indicated that the
218
WW domain mediated the direct interaction between WWP2 and Paip1 (Fig. 3D). The C2
219
or HECT domains of WWP2 did not show the same regulatory action. The
220
abovementioned results contradicted a previous finding, which stated that the WW
221
domains of Nedd4 family members preferred to recruit substrates (29). Paip1 comprised
222
two binding sites for PABP, namely, PAM1 and PAM2 (15). PAM2 was required for the
223
binding of Paip1 to eIF3. Different parts of Paip1 were fused to GST to identify the
224
WWP2-binding site in Paip1. His-WWP2 was detected in reactions involving the
225
full-length protein, the fusion protein with retained Paip1 N terminus (comprising amino 11
226
acids 1 to 143), and the PAM2 motif (comprising amino acids 116 to 143). However, the
227
large C-terminal region (comprising amino acids 144 to 479) was not required for the
228
binding of Paip1 to WWP2 (Fig. 3E). The PAM2 motif was previously identified in
229
numerous proteins with diverse functions (30), thereby suggesting that PAM2
230
participated in protein–protein interactions in a wide range of cellular processes. Two of
231
the three PxxY motifs were typical binding regions of the WW domains of the Nedd4
232
family members (31) and were located in the PAM2 motif of Paip1 (Fig. 1B). Paip1
233
interacted with PABP via the PAM2 motif. We measured the interaction of Paip1 and
234
PABP upon WWP2 silencing and overexpression to determine whether or not WWP2 and
235
PABP competed for Paip1 binding. WWP2 did not affect the interaction of Paip1 and
236
PABP (Fig. 3F), thereby suggesting that Paip1’s PAM2 motif (comprising amino acids
237
116 to 143) sufficiently interacted with the WW domain of WWP2.
238
Degradation and ubiquitination of Paip1 by WWP2 depends on the presence of
239
the PEFYPSGY sequence. Based on data from previous studies, we speculated that the
240
two PxxY motifs (PEFYPSGY sequence) in PAM2 participated in WWP2’s recognition
241
and degradation of Paip1. We transfected different Paip1 deletion mutants into HEK293T
242
cells with Flag-WWP2-WT to determine the importance of the two PxxY motifs.
243
Degradation analysis showed that mutants 144 to 479 included one PxxY motif that was
244
not degraded by overexpressed WWP2. By contrast, the N-terminal mutants 1 to 143,
245
which comprised the first two PxxY motifs, were efficiently degraded by WWP2 (Fig.
246
4A). This phenomenon suggested that the critical degradation signal was present in the 12
247
mutants. To further determine which PxxY motif was critical for WWP2-mediated
248
degradation, we used the ΔPEFY, ΔPSGY, and ΔPEFYPSGY forms of Paip1 to analyze
249
protein levels in the presence or absence of WWP2. Paip1-ΔPEFY and Paip1-ΔPSGY
250
were both downregulated by WWP2. However, WWP2 did not downregulate
251
Paip1-ΔPEFYPSGY (Fig. 4B). The two PxxY motifs and the PEFYPSGY sequence were
252
necessary for WWP2 to recognize Paip1.
253
We performed an ubiquitination assay to test whether or not the resistance of the
254
mutant Paip1-ÄPEFYPSGY to WWP2-mediated degradation was caused by abrogated
255
ubiquitination. WWP2 did not ubiquitylate the truncate (Fig. 4C). The PEFYPSGY
256
sequence was critical in the catalytic activity of WWP2 during Paip1 ubiquitination and
257
degradation.
258
WWP2 negatively regulates Paip1-mediated translation enhancement. To gain
259
insight into the functional relationship between WWP2 and Paip1, we initially determined
260
the translational stimulatory activity via in vivo translation assays. Under the control of
261
the Tet-off promoter expressing WWP2-WT or WWP2-C838 A, DNA vectors were
262
transfected into HeLa cells along with constructs expressing Renilla luciferase and tTA.
263
Each construct yielded comparable amounts of protein. Paip1 was absent in the extract
264
expressing WWP2 (Fig. 5A). The relative induction of luciferase activity was determined
265
by calculating the ratio of Renilla luciferase activity between induced (without Tet) and
266
repressed (with Tet at 300 ng/ml) expressions of WWP2. Luciferase activity was lower
267
with the overexpression of the wild-type WWP2 plasmid than with the expression of the 13
268
vector alone (Fig. 5B lanes 1, 2, 3, 4, and 5). However, the overexpression of
269
WWP2-C838 A did not affect in vivo translation (Fig. 5B lanes 6 and 7), thereby
270
suggesting that the ligase activity of WWP2 was required. WWP2 overexpression did not
271
affect Renilla luciferase mRNA levels (Fig. 5C).
272
To determine whether or not the translational suppression induced by WWP2
273
depended on the presence of Paip1, we examined the effect of Paip1 silencing via siRNA
274
on WWP2 activity. The expressions of WWP2 and Paip1 were confirmed by Western
275
blot analysis (Fig. 5D). No significant translational repression was observed when Paip1
276
was knocked down compared with the control (Fig. 5E). Renilla luciferase mRNA levels
277
were not affected by siRNA treatments (Fig. 5F). Therefore, WWP2 repressed mRNA
278
translation depending on the presence of Paip1. These data confirmed the importance of
279
the protein level regulation of Paip1 by WWP2. We demonstrated that WWP2 is a novel
280
regulator of translational initiation.
281 282
Discussion
283
Translation is important in the regulation of gene expression and is implicated in the
284
control of cell growth, proliferation, and differentiation (32–34). In eukaryotes, initiation
285
is the rate-limiting step of translation under most circumstances; initiation is a major
286
target for regulation (33). Paip1 is a mammalian PABP that binds to eIF4A and eIF3 and
287
stimulates translational initiation. In the present study, we showed that Paip1 protein was
288
degraded by HECT ubiquitin ligase WWP2. The following findings from the present 14
289
study directly corroborate the use of Paip1 as a physiological substrate of WWP2. WWP2
290
directly interacted with Paip1, and the interaction depended on the integrity of the WW
291
domain of WWP2. The loss of WWP2 impeded Paip1 turnover. WWP2 promoted Paip1
292
ubiquitination both in vivo and in a reconstituted in vitro system. The ubiquitin ligase
293
activity of WWP2 and the PxxY motif of Paip1 were critical for ubiquitination and
294
degradation. Previous studies have demonstrated the involvement of WWP2 in the
295
regulation of transcription, embryonic stem cell development, cellular transport, T-cell
296
activation processes, and tumorigenesis by targeting distinct substrates (35). In the
297
present study, we revealed that Paip1 was a novel substrate of WWP2. We also
298
emphasized the importance of WWP2 in the regulation of translation.
299
The involvement of deregulation of translational initiation in cancer development and
300
progression became the focus of various studies only recently. WWP2 mediates the
301
depletion of phosphatase and tensin homolog and consequently elevates
302
phosphoinositide-3-kinase–protein kinase B pathway activity (36). Such regulatory
303
activities may enhance eIF4E function and activate cap-dependent translation initiation,
304
leading to the selective increase of translation of key mRNAs; these mRNAs are involved
305
in tumor growth, angiogenesis, and cell survival (37). Thus, we hypothesized that
306
WWP2-mediated degradation of Paip1 selectively reduced the expression of numerous
307
potent growth and survival factors. The role of WWP2 in tumorigenesis, which depends
308
on WWP2’s regulation of translation, requires further investigation.
309
Circularization of the mRNA by bridging the mRNA 5’ and 3’ is an important step in 15
310
the process of translation initiation. In yeast, eIF4G interacts with PABP to contribute to
311
the circularization of the mRNA and mediation of the poly(A) tail-dependent translation
312
(11). In plants, PABP binds to eIF-iso4G and eIF4B, thereby increasing the RNA-binding
313
activity of PABP (13). However, mammalian cells possess dual systems, namely,
314
PABP–Paip1 and PABP–eIF4G. These systems regulate the formation of mRNP. The
315
presence of Paip1 in animal cells reflects evolutionary advancement and allows higher
316
eukaryotes to link PABP to eIF4A function. Information is lacking on whether the
317
degradation of Paip1 by WWP2 reduces the rate of the circular mRNA conformation and
318
inhibits mRNA translation. Paip1’s sequence and function are similar to those of the
319
eIF4G protein. WWP2 may also regulate the protein level of eIF4G in human cells, but
320
the relationship between WWP2 and eIF4G requires further study.
321
The stimulation of translation by Paip1 in vivo decreased upon the deletion of the
322
N-terminal sequence containing PABP and the eIF3 binding domain known as PAM2
323
(22). In the present study, PAM2 was critical for the binding of PABP to WWP2.
324
Paip1-interacting proteins can potentially compete for Paip1 binding via the PAM2
325
domain, thereby regulating translation. Further studies are required to determine the
326
presence of an overlap among the binding sites of WWP2, eIF3, and PABP in Paip1.
327
WWP2 was identified as a typical ubiquitin ligase for Paip1. We showed that the
328
overexpression of WWP2 significantly decreased Paip1 protein level, and such decrease
329
suppressed translation. We also discovered that the WW domain of WWP2 interacted
330
with Paip1 fragments, which contained two PxxY motifs. The degradation and 16
331
ubiquitination of Paip1 by WWP2 was transiently abolished by the deletion of the two
332
PxxY motifs. WWP2 is an important translational suppressor that participates in Paip1
333
degradation. The function of WWP2 in the regulation of translation was clarified for the
334
first time. The mechanisms underlying WWP2’s regulation of Paip1 should be studied
335
further to reveal the details of WWP2’s function.
336 337
ACKNOWLEDGMENTS
338
This work was supported by grants from the Nature Science of Shandong Province,
339
Q2008C08. The funders had no role in the study design, data collection, data analysis,
340
decision to publish, or preparation of the manuscript.
341 342
REFERENCES
343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359
1.
Parsyan A, Hernandez G, Meterissian S. 2012. Translation initiation in colorectal cancer. Cancer Metastasis Rev 31:387-395.
2.
Jia Y, Polunovsky V, Bitterman PB, Wagner CR. 2012. Cap-dependent translation initiation factor eIF4E: an emerging anticancer drug target. Med Res Rev 32:786-814.
3.
Graff JR, Konicek BW, Carter JH, Marcusson EG. 2008. Targeting the eukaryotic translation initiation factor 4E for cancer therapy. Cancer research 68:631-634.
4.
Cheung YN, Maag D, Mitchell SF, Fekete CA, Algire MA, Takacs JE, Shirokikh N, Pestova T, Lorsch JR, Hinnebusch AG. 2007. Dissociation of eIF1 from the 40S ribosomal subunit is a key step in start codon selection in vivo. Genes Dev 21:1217-1230.
5.
Chiu WL, Wagner S, Herrmannova A, Burela L, Zhang F, Saini AK, Valasek L, Hinnebusch AG. 2010. The C-terminal region of eukaryotic translation initiation factor 3a (eIF3a) promotes mRNA recruitment, scanning, and, together with eIF3j and the eIF3b RNA recognition motif, selection of AUG start codons. Molecular and cellular biology 30:4415-4434.
6.
Pisarev AV, Unbehaun A, Hellen CU, Pestova TV. 2007. Assembly and analysis of eukaryotic translation initiation complexes. Methods Enzymol 430:147-177.
7.
Lorsch JR, Dever TE. 2010. Molecular view of 43 S complex formation and start site selection in eukaryotic translation initiation. THE JOURNAL OF BIOLOGICAL CHEMISTRY 285:21203-21207. 17
360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400
8.
Preiss T, M WH. 2003. Starting the protein synthesis machine: eukaryotic translation initiation. Bioessays 25:1201-1211.
9.
Prevot D, Darlix JL, Ohlmann T. 2003. Conducting the initiation of protein synthesis: the role of eIF4G. Biol Cell 95:141-156.
10.
Kessler SH, Sachs AB. 1998. RNA recognition motif 2 of yeast Pab1p is required for its functional interaction with eukaryotic translation initiation factor 4G. Molecular and cellular biology 18:51-57.
11.
Tarun SZ, Jr., Sachs AB. 1996. Association of the yeast poly(A) tail binding protein with translation initiation factor eIF-4G. The EMBO Journal 15:7168-7177.
12.
Tarun SZ, Jr., Wells SE, Deardorff JA, Sachs AB. 1997. Translation initiation factor eIF4G mediates in vitro poly(A) tail-dependent translation. Proceedings of the National Academy of Sciences of the United States of America 94:9046-9051.
13.
Le H, Tanguay RL, Balasta ML, Wei CC, Browning KS, Metz AM, Goss DJ, Gallie DR. 1997. Translation initiation factors eIF-iso4G and eIF-4B interact with the poly(A)-binding protein and increase its RNA binding activity. THE JOURNAL OF BIOLOGICAL CHEMISTRY 272:16247-16255.
14.
Khaleghpour K, Kahvejian A, De Crescenzo G, Roy G, Svitkin YV, Imataka H, O'Connor-McCourt M, Sonenberg N. 2001. Dual interactions of the translational repressor Paip2 with poly(A) binding protein. Molecular and cellular biology 21:5200-5213.
15.
Roy G, De Crescenzo G, Khaleghpour K, Kahvejian A, O'Connor-McCourt M, Sonenberg N. 2002. Paip1 interacts with poly(A) binding protein through two independent binding motifs. Mol Cell Biol 22:3769-3782.
16.
Imataka H, Olsen HS, Sonenberg N. 1997. A new translational regulator with homology to eukaryotic translation initiation factor 4G. The EMBO Journal 16:817-825.
17.
Levy-Strumpf N, Deiss LP, Berissi H, Kimchi A. 1997. DAP-5, a novel homolog of eukaryotic translation initiation factor 4G isolated as a putative modulator of gamma interferon-induced programmed cell death. Molecular and cellular biology 17:1615-1625.
18.
Yamanaka S, Poksay KS, Arnold KS, Innerarity TL. 1997. A novel translational repressor mRNA is edited extensively in livers containing tumors caused by the transgene expression of the apoB mRNA-editing enzyme. Genes Dev 11:321-333.
19.
Morino S, Imataka H, Svitkin YV, Pestova TV, Sonenberg N. 2000. Eukaryotic translation initiation factor 4E (eIF4E) binding site and the middle one-third of eIF4GI constitute the core domain for cap-dependent translation, and the C-terminal one-third functions as a modulatory region. Molecular and cellular biology 20:468-477.
20.
Imataka H, Sonenberg N. 1997. Human eukaryotic translation initiation factor 4G (eIF4G) possesses two separate and independent binding sites for eIF4A. Molecular and cellular biology 17:6940-6947.
21.
Craig AW, Haghighat A, Yu AT, Sonenberg N. 1998. Interaction of polyadenylate-binding protein with the eIF4G homologue PAIP enhances translation. Nature 392:520-523.
22.
Martineau Y, Derry MC, Wang X, Yanagiya A, Berlanga JJ, Shyu AB, Imataka H, Gehring K, Sonenberg N. 2008. Poly(A)-binding protein-interacting protein 1 binds to eukaryotic translation initiation factor 3 to stimulate translation. Mol Cell Biol 28:6658-6667. 18
401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437
23.
Martineau Y, Wang X, Alain T, Petroulakis E, Shahbazian D, Fabre B, Bousquet-Dubouch MP, Monsarrat B, Pyronnet S, Sonenberg N. 2014. Control of Paip1-eukayrotic translation initiation factor 3 interaction by amino acids through S6 kinase. Mol Cell Biol 34:1046-1053.
24.
Zou W, Chen X, Shim JH, Huang Z, Brady N, Hu D, Drapp R, Sigrist K, Glimcher LH, Jones D. 2011. The E3 ubiquitin ligase Wwp2 regulates craniofacial development through mono-ubiquitylation of Goosecoid. Nature Cell Biology 13:59-65.
25.
Nakamura Y, Yamamoto K, He X, Otsuki B, Kim Y, Murao H, Soeda T, Tsumaki N, Deng JM, Zhang Z, Behringer RR, Crombrugghe B, Postlethwait JH, Warman ML, Nakamura T, Akiyama H. 2011. Wwp2 is essential for palatogenesis mediated by the interaction between Sox9 and mediator subunit 25. Nature Communications 2:251.
26.
Maddika S, Kavela S, Rani N, Palicharla VR, Pokorny JL, Sarkaria JN, Chen J. 2011. WWP2 is an E3 ubiquitin ligase for PTEN. Nature Cell Biology 13:728-733.
27.
Soond SM, Chantry A. 2011. Selective targeting of activating and inhibitory Smads by distinct WWP2 ubiquitin ligase isoforms differentially modulates TGFbeta signalling and EMT. Oncogene 30:2451-2462.
28.
Gossen M, Bujard H. 1992. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proceedings of the National Academy of Sciences of the United States of America 89:5547-5551.
29.
Bernassola F, Karin M, Ciechanover A, Melino G. 2008. The HECT family of E3 ubiquitin ligases: multiple players in cancer development. Cancer Cell 14:10-21.
30.
Albrecht M, Lengauer T. 2004. Survey on the PABC recognition motif PAM2. Biochem Biophys Res Commun 316:129-138.
31.
Ingham RJ, Gish G, Pawson T. 2004. The Nedd4 family of E3 ubiquitin ligases: functional diversity within a common modular architecture. Oncogene 23:1972-1984.
32. 33.
Conlon I, Raff M. 1999. Size control in animal development. Cell 96:235-244. Gingras AC, Raught B, Sonenberg N. 1999. eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation. Annu Rev Biochem 68:913-963.
34.
Miron M, Verdu J, Lachance PE, Birnbaum MJ, Lasko PF, Sonenberg N. 2001. The translational inhibitor 4E-BP is an effector of PI(3)K/Akt signalling and cell growth in Drosophila. Nature Cell Biology 3:596-601.
35.
Chen A, Gao B, Zhang J, McEwen T, Ye SQ, Zhang D, Fang D. 2009. The HECT-type E3 ubiquitin ligase AIP2 inhibits activation-induced T-cell death by catalyzing EGR2 ubiquitination. Molecular and cellular biology 29:5348-5356.
36.
Chantry A. 2011. WWP2 ubiquitin ligase and its isoforms: new biological insight and promising disease targets. Cell cycle 10:2437-2439.
37.
Graff JR, Konicek BW, Carter JH, Marcusson EG. 2008. Targeting the eukaryotic translation initiation factor 4E for cancer therapy. Cancer Res 68:631-634.
438 439 19
440
FIGURE LEGENDS
441
Fig. 1. WWP2 negatively regulates Paip1 protein level. (A) MG132 treatment upregulates
442
Paip1 protein level. HEK293T cells were treated with MG132 (20 µM) for 8 h before
443
harvest. Endogenous Paip1, PABP, and Paip2 levels were analyzed by immunoblotting.
444
GAPDH was used as an internal control. (B) Schematic of Paip1 protein. The positions of
445
PxxY motifs are labeled. (C) WWP2 specifically reduces Paip1 protein amount. The
446
indicated Nedd4 family of E3 ligases was co-transfected with Paip1 into HEK293T cells,
447
and cell lysates were analyzed by immunoblotting. (D) WWP2 downregulates Paip1 in a
448
proteasome-dependent manner. Increasing concentrations of Flag-WWP2 plasmids were
449
co-transfected into HEK293T cells with Myc-Paip1. Cells were treated with MG132
450
(20 μM) and lactacystin (30 μM) or dimethyl sulfoxide (DMSO) for 16 h. The cell lysates
451
were analyzed by immunoblotting. (E) WWP2 decreases the Paip1 protein level in a
452
dose-dependent manner. Cells were transfected with increasing amounts of WWP2-WT
453
or the catalytic mutant form WWP2-C838A. Subsequently, Paip1 level was detected. (F)
454
WWP2 can not affect the mRNA level of Paip1. Paip1 mRNA prepared from the
455
transfected HEK293T cells was analyzed by real-time PCR assay. Data are presented as
456
mean ± S.D. (n = 3). (G) WWP2 depletion increases the endogenous Paip1 protein level.
457
HEK293T and HeLa cells were transfected with non-targeted control or with
458
WWP2-specific siRNA. The endogenous WWP2 and Paip1 levels were analyzed by
459
Western blot. (H) WWP2 depletion can not affect the Paip1 mRNA level. Paip1 mRNA
460
prepared from the transfected HEK293T and HeLa cells was analyzed by real-time PCR 20
461
assay. Data are presented as mean ± S. D. (n = 3). (I) WWP2 can not affect the
462
endogenous Paip2 and PABP protein levels. Cells were transfected with the WWP2-WT
463
or the catalytic mutant form WWP2-C838A. Paip2 and PABP protein levels were
464
detected by immunoblotting (top). HEK293T cells were transfected with non-targeted
465
control or WWP2–specific siRNA. The endogenous WWP2 and PABP protein levels
466
were analyzed by immunoblotting (bottom). (J) Overexpression of Paip2 can not affect
467
WWP2-mediated degradation of Paip1. HEK293T cells were transfected with the vector
468
or with Paip2. At 24 h after transfection, cells were transfected with WWP2 and analyzed
469
by immunoblotting.
470
Fig. 2. WWP2 promotes the ubiquitination and degradation of Paip1. (A) WWP2 reduces
471
the half-life of endogenous Paip1 protein. HEK293T cells were transfected with control
472
plasmid or Flag-WWP2-WT or the catalytic mutant form Flag-WWP2-C838 A, and cells
473
were treated with the protein synthesis inhibitor CHX (10 µg/ml) for the indicated
474
durations before harvest. Paip1 protein expression was analyzed. Quantitative analysis
475
was performed by measuring integrated optical density using the program Gel-Pro
476
analyzer. Data are presented as mean ± S. D. (n = 3). (B) Depletion of WWP2 prolonges
477
the half-life of endogenous Paip1 protein. HEK293T cells were transfected with control
478
siRNA or WWP2 siRNA and treated with CHX. Paip1 protein expression was
479
subsequently analyzed. Quantitative analysis was performed by measuring integrated
480
optical density using the program Gel-Pro analyzer. Data are presented as mean ± S. D. (n
481
= 3). (C) WWP2 catalyzes the ubiquitination of Paip1 in vitro. A mixture comprising 21
482
purified HA-ubiquitin, E1, E2 (UbcH5c), bacteria-expressed and purified
483
His-WWP2-WT or C838A, and GST-Paip1 or GST was used for in vitro ubiquitination
484
assays and subsequent immunoblotting with anti-HA. (D) WWP2 enhances the
485
ubiquitination of Paip1 in vivo. HEK293T cells were transfected with HA-Ub,
486
Myc-Paip1, control vector, or Flag-WWP2, and treated with MG132 as indicated.
487
Ubiquitinated Paip1 was immunoprecipitated by anti-Myc antibody and analyzed by
488
immunoblotting. (E) WW and HECT domains of WWP2 were involved in the
489
ubiquitination of Paip1. HA-Ub, Myc-Paip1, and WWP2 constructs were co-transfected
490
into HEK293T cells and treated with MG132. Ubiquitinated Paip1 was
491
immunoprecipitated with anti-HA antibody and analyzed by immunoblotting.
492
Fig. 3. Mapping of the interaction region between the truncated domains of WWP2 and
493
Paip1. (A) WWP2 was colocalized with Paip1 within the cytoplasm. Myc-Paip1 and
494
Flag-WWP2 were co-transfected into HEK293T cells, and cells were stained 24 h later
495
using mouse anti-Flag and rabbit anti-Myc antibodies for visualization by confocal
496
microscopy. (B) Paip1 was co-immunoprecipitated with wild type and catalytic mutant
497
forms of WWP2. Wild type and catalytic mutant forms of WWP2 and Myc-Paip1 were
498
transfected into HEK293T cells. Cell lysates were immunoprecipitated with anti-Flag
499
antibody. Lysates and immunoprecipitates were analyzed by immunoblotting. (C)
500
Immunoprecipitation analysis of WWP2 and Paip1 in vivo. HeLa cells were transfected
501
with negative control siRNA or siRNA targeted Paip1, and cells were harvested after 36 h.
502
The extracts were immunoprecipitated with anti-WWP2 antibody and subsequently 22
503
analyzed by immunoblotting using anti-WWP2, anti-Paip1, anti-eIF3a, and anti-PABP
504
antibodies. Pre-immune IgG was used as the control. (D) Direct interaction between the
505
WW domain of WWP2 and Paip1. GST pull-downs were conducted with GST or
506
GST-WWP2 fragments along with purified His-Paip1. Input and pull-down samples were
507
subjected to immunoblotting with anti-GST and anti-His antibodies. Input represented 10%
508
of the sample used for the pull-down. (E) PAM2 motif of Paip1 showed direct binding to
509
WWP2. GST pull-downs were performed with GST or GST-Paip1 fragments along with
510
purified His-WWP2. Input and pull-down samples were subjected to immunoblotting
511
with anti-GST and anti-His antibodies. Input represented 10% of the sample used for
512
pull-down. (F) WWP2 can not affect the interaction between Paip1 and PABP. HEK293T
513
cells were transfected with Flag-WWP2-WT or the catalytic mutant form
514
Flag-WWP2-C838A. Cell lysates were immunoprecipitated with anti-Paip1 antibody.
515
Both the lysate and immunoprecipitates were analyzed by immunoblotting (left).
516
HEK293T cells were transfected with control siRNA or WWP2 siRNA. Cell lysates were
517
immunoprecipitated with anti-Paip1 antibody. Lysates and immunoprecipitates were
518
analyzed by immunoblotting (right).
519
Fig. 4. PEFYPSGY sequence is sufficient for the ubiquitination and degradation of Paip1.
520
(A) HEK293T cells were transfected with plasmids expressing either full-length Paip1 or
521
mutants of Paip1 with or without a plasmid encoding Flag-WWP2-WT. (B) PEFYPSGY
522
sequence maintains the WWP2-mediated degradation of Paip1. HEK293T cells were
523
transfected with plasmids expressing Paip1-ΔPEFY, Paip1-ΔPSGY, or 23
524
Paip1-ΔPEFYPSGY with or without Flag-WWP2. The cell lysates were analyzed by
525
immunoblotting. (C) PEFYPSGY sequence attenuated WWP2-mediated ubiquitination of
526
Paip1. HEK293T cells were co-transfected with Myc-Paip1 deletions and HA-Ub with or
527
without Flag-WWP2. Ubiquitinated Paip1 was immunoprecipitated with an anti-Myc
528
antibody and protein A/G-agarose beads under denaturing conditions to eliminate any
529
WWP2-associated protein by non-covalent bonding.
530
Fig. 5. Overexpression of WWP2 represses translation via the Paip1 protein. (A) Cells
531
were transfected with the indicated pTet-HA-WWP2-WT or catalytic mutant form
532
pTet-HA-WWP2-C838A plasmids along with constructs expressing Renilla luciferase
533
and tTA. Cells were cultured in a medium containing 0 or 300 ng/ml of Tet. Cells were
534
harvested and subjected to Western blot analysis with the indicated antibodies. (B)
535
Renilla luciferase activity was quantified in HeLa cell extracts harvested in (A) and
536
normalized on the total protein level. Relative induction of the luciferase reporter was
537
determined by calculating the ratio of Renilla luciferase activity between induced
538
(without Tet) and repressed (with 300 ng/ml Tet) expression of the indicated HA-WWP2.
539
Error bars denote the standard error of the mean for the three independent experiments.
540
(C) Quantitative real-time PCR of Renilla luciferase mRNA obtained from total RNA of
541
duplicate HeLa cells harvested in (A). Data are presented as mean ± S. D. (n = 3). (D)
542
HeLa cells were transfected with the indicated siRNA sequences. At 24 h after siRNA
543
transfection, cells were transfected as described in (A). Cells were subsequently placed in
544
a medium containing 0 or 300 ng/ml of Tet. Cells were harvested and subjected to 24
545
Western blot analysis with the indicated antibodies. (E) Renilla luciferase activity was
546
quantified in HeLa cell extracts harvested in (D) and normalized on the total protein level.
547
Relative induction of the luciferase reporter was determined by calculating the ratio of
548
Renilla luciferase activity between induced (without Tet) and repressed (with 300 ng/ml
549
Tet) expression of the indicated HA-WWP2. Error bars denote the standard error of the
550
mean for three independent experiments. (F) Quantitative RT–PCR of Renilla luciferase
551
mRNA obtained from the total RNA of the duplicate HeLa cells harvested in (D). Data
552
are presented as mean ± S. D. (n = 3).
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